Standard compound for immunoassay for dioxin and method of immunoassay for dioxin

ABSTRACT

A compound of formula (1) and an immunoassay method for quantitative determination of dioxins in a sample using as a standard the compound of the following formula (1): 
                         
wherein R 1 , R 2 , R 3  and R 4  may be the same or different and represent chlorine or hydrogen, n is an integer from 1 to 10, and Z represents an amino acid residue or peptide.

RELATED APPLICATIONS

This is a U.S. national phase filing under 35 U.S.C. § 371 ofPCT/JP03/10394 filed Aug. 18, 2003 and claims priority from JP2002-251616 filed Aug. 29, 2002.

TECHNICAL FIELD

The present invention relates to an immunoassay standard for dioxins.The present invention further relates to an immunoassay method fordioxins using this standard, and more specifically to a method ofcomputing the concentration or toxic equivalent (TEQ) of dioxins in theair, exhaust gases, soils, rivers, combustion ashes and the like using adioxin analogue as a reference.

BACKGROUND ART

Dioxins is a general term for polychlorinated dibenzo-p-dioxins (PCDDs),polychlorinated dibenzofurans (PCDFs), and coplanar polychlorinatedbiphenyls (Co-PCBs). A great number of isomers that differ in thechlorine substitution patterns exist for these three types of skeletalstructures. Among the isomers of PCDDs and PCDFs, those having chlorinesubstituents at the 2-, 3-, 7-, and 8-positions are highly toxic andknown to cause dermatitis, multiple neurosis, nystagmus, hepaticinsufficiency and like symptoms due to the chlorine substitution.

It is also known that long-term exposure to dioxins even in lowconcentrations can cause chronic symptoms such as porphyria cutaneatarda and also exhibit various toxic properties such as teratogenicityand carcinogenicity.

Furthermore, in recent years, so-called “endocrine disruptors” havingthe action of disrupting endocrine functions in humans and wild animalshave been the focus of attention as a global environmental problem. Ithas also been revealed that dioxins might be an endocrine disruptorhaving estrogen activity.

It has become clear that dioxins with their various toxic properties arecontained in chemicals such as herbicides and insecticides, exhaustgases and fly ashes from garbage incineration facilities, waste waterfrom papermills, etc. Dioxins are thus detected not only inenvironmental samples from the air, soils, waters and sediments ofrivers, harbors and ports around big cities, etc. but also in biologicalsamples such as foods, blood, urine and mothers' milk. Since such awidespread contamination of the environment has been a big socialproblem, there is a pressing need to know the amount of dioxin exposurein the environment.

The measurement of dioxins requires high-precision analytical data.Therefore, official methods that comprise extracting, concentrating andpurifying dioxins using various chromatographic techniques andsubsequent analysis using an expensive analyzer such as a highresolution gas chromatography/mass spectrometer are conventionally used.Such analytical methods are highly sensitive and capable ofmulti-component analysis so that two or more compounds can be identifiedand quantitatively determined at one time. On the other hand, suchmethods have problems in that capital investment such as expensivespecial equipment and clean rooms is necessary and specialists who areskilled in analysis are required and it takes a long time to obtainresults because of the complicated pretreatment.

For such reasons, the development of a highly sensitive, easy dioxinimmunoassay method is desired. Environmental pollutant detectiontechniques using an antibody immunoassay have attracted attention toachieve this object.

Immunoassay is a method for detecting or quantitatively determining atrace amount of an antigen using the capacity of an antibody tospecifically recognize the antigen, whereby the antigen can be measuredwith high sensitivity due to the high affinity and high specificity ofthe antibody for the antigen. Thus immunoassay has various merits suchas simplicity in pretreatment of samples, easy and quick measurement ofmultiple samples and low cost per measurement, and has been used in awide variety of fields such as medicine, biochemistry, pharmaceuticalsciences and agriculture. To detect or quantitatively determine thetargeted substance by immunoassay, it is necessary to label the antibodyor antigen, and various labeling methods have been developed. Due to itssimplicity, enzyme immunoassay (EIA) using an enzyme has widely beenused in clinical tests and the biochemistry field to quantitativelydetermine targeted substances in biological samples. EIA can be roughlyclassified into competitive assays and non-competitive assays, based onthe form of antigen-antibody interaction. Low molecular weight compoundssuch as dioxins are usually determined by competitive assays.

In EIA, the concentration of a targeted compound in a sample iscalculated from a standard curve that is prepared by using the samecompound as the targeted compound as a standard and quantitativelydetermining the standard in the same manner as the sample. However,since dioxins encompass isomers of three types of compounds havingdifferent skeletal structures, the isomers being different in thechlorine substitution pattern, it is difficult to decide which compoundshould be used as a standard.

Since the toxicity of dioxins differs among congeners and isomers, thetoxicity level of dioxins, a mixture of congeners and isomers havingdifferent toxicities, varies depending on the ratio of the constituentisomers. A simple sum of the individual isomer amounts does notaccurately represent dioxin toxicity.

Many of the dioxin EIA systems so far developed utilize the most toxic2,3,7,8-tetrachlorobenzo-p-dioxin (2,3,7,8-TeCDD) as a standard (Anal.Chem. 70, 1092–1099). The toxicity of individual dioxin isomers isexpressed by toxic equivalency factors (TEFs), which are relativetoxicity factors of the isomers compared to 2,3,7,8-TeCDD which is setas 1. The concentrations of the individual isomers are multiplied bytheir TEFs to calculate their toxicity. The sum of these toxicity valuesis the toxic equivalent (TEQ), i.e., the total toxicity of all theisomers existing in the target.

Thus the EIA system, which mainly measures 2,3,7,8-TeCDD, can not besaid to be an accurate measurement system for dioxin toxicity. Inparticular, dioxin TEQ of exhaust gas emitted from waste incinerators,which are a main source of dioxins, is known to be highly correlated tothe concentration of pentachlorinated dibenzofurans or hexachlorlnateddibenzofurans rather than 2,3,7,8-TeCDD. In addition, when the analytesare exhaust gas samples, there are cases in which the measurementsobtained using the conventional EIA system are significantly differentfrom the results of instrumental analysis. Thus the use of the EIAsystem may be limited.

Moreover, when 2,3,7,8-TeCDD or like dioxin isomers are used as astandard in EIA, the assayer should handle a highly toxic compound forthe standard preparation, resulting in the necessity of ensuring safetyand involving problems such as the mental burden on the assayer.

Therefore, a non-toxic compound is desired as a standard for thequantitative determination of dioxins in samples. In this connection,Japanese Unexamined Patent Publications Nos. 128731/2002, 131316/2002and 155023/2002 describe using a chlorophenol derivative as a standard.

DISCLOSURE OF THE INVENTION

An object of the invention is to provide a dioxin immunoassay standardthat does not have a toxic equivalency factor (TEF). Another object ofthe invention is to provide an immunoassay method using this standardthat enables simple, highly sensitive measurement of dioxinconcentration or toxic equivalent in environmental samples.

To achieve the above objects, the present inventors carried outintensive research and found the following:

-   (i) When the concentration of dioxins in a sample is determined by    immunoassay using as a standard a chlorophenol derivative of formula    (1)

wherein R¹, R², R³ and R⁴ may be the same or different and representchlorine or hydrogen, n is an integer from 1 to 10, and Z represents anamino acid residue or peptide, and the TEQ is calculated from theconcentration, there is a high correlation between the obtained TEQ andthat obtained by the official method of determining the concentration ofeach dioxin in a sample.

-   (ii) Since the above chlorophenol derivative does not have a toxic    equivalency factor (TEF), the use of this compound as a dioxin assay    standard enables safe calculation of dioxin concentration and toxic    equivalent (TEQ) of environmental samples.

The present invention has been accomplished on the basis of the abovefindings. The invention provides the following:

1. A compound of formula (1)

wherein R¹, R², R³ and R⁴ may be the same or different and representchlorine or hydrogen, n is an integer from 1 to 10, and Z represents anamino acid residue or peptide.

2. An immunoassay standard for dioxins comprising a compound of formula(1)

wherein R¹, R², R³ and R⁴ may be the same or different and representchlorine or hydrogen, n is an integer from 1 to 10, and Z represents anamino acid residue or peptide.

3. An immunoassay kit for dioxins comprising the compound of item 1.

4. A kit according to item 3, further comprising an anti-dioxinsantibody.

5. A kit according to item 3 or 4, further comprising a competitiveantigen.

6. An immunoassay method for quantitative determination of dioxins, themethod using the compound of item 1 as a standard.

7. An immunoassay method according to item 6 selected from the groupconsisting of enzyme immunoassay, radioactive immunoassay andfluoroimmunoassay.

8. An immunoassay method for determining the dioxins concentration of asample and calculating the TEQ, the method using the compound of item 1as a standard.

9. An immunoassay method according to claim 8 selected from the groupconsisting of enzyme immunoassay, radioactive immunoassay andfluoroimmunoassay.

10. An immunoassay method for dioxins comprising the following steps:

-   (1) reacting a sample with an anti-dioxin antibody to determine the    amount of antigen reacted with the antibody; and-   (2) comparing the amount of antigen reacted with the antibody in    step (1) with that determined by allowing a known concentration of    the compound of item 1 to react with the anti-dioxin antibody to    compute the dioxins concentration in the sample.

11. An immunoassay method according to item 10 wherein the amount ofantigen reacted with the antibody is determined by a method selectedfrom enzyme immunoassay. radioactive immunoassay and fluoroimmunoassay.

The present invention provides an immunoassay standard for dioxins thatdoes not have a toxic equivalency factor (TEF), and an immunoassaymethod using this standard that enables simple, highly sensitivemeasurement of dioxins concentration or toxic equivalent ofenvironmental samples.

More specifically, when the dioxin concentration of a sample isquantitatively determined using the immunoassay standard of formula (1)and the TEQ is calculated therefrom, the concentration and TEQ have agood correlation with the TEQ obtained by the official method ofdetermining the concentrations of dioxins having TEFs by instrumentalanalysis. The use of the compound of the invention as a standard forquantitative determination of dioxins enables accurate, highly sensitiveTEQ determination of samples, allowing an accurate evaluation of dioxintoxicity compared to EIA in the prior art.

In addition, the hitherto known official method takes a long time toevaluate TEQ because TEQ is calculated by measuring the concentrationsof 29 types of dioxins and computing TEQ from the concentrations. Incontrast, immunoassay using the compound of the invention takes acomparatively short time to evaluate TEQ.

The standard of the invention is a compound to which the WHO has notassigned any toxic equivalency factor, thus being assumed to be anon-toxic compound. The non-toxicity is also clear from the fact thatthe compound of the invention is a derivative obtained by the additionof a methylene chain and a peptide to a commercially availablechlorophenol compound (for example, 2,4,5-trichlorophenol). The use ofthe compound of the invention can therefore greatly enhance safety indioxin immunoassay.

As shown above, the compound and the immunoassay method of the inventionfind wide application in environmental analysis and the like and arealso useful in the analysis of biological samples such as foods,mothers' milks, blood and urine.

DISCLOSURE OF THE INVENTION

The present invention will be described below in detail.

(I) Compound of the Invention

Basic Construction

The compound of the following formula (1) is a novel compound notdescribed in any publication:

wherein R¹, R², R³ and R⁴ may be the same or different and representchlorine or hydrogen, n is an integer from 1 to 10, and Z represents anamino acid residue or peptide.Preferable Compounds

Although the compound of formula (1) is not particularly limited in thenumber and position of chlorine substituents, it is preferable, in viewof the reactivity with anti-dioxin antibodies, that the total number ofchlorine substituents in the compound is 3 or more, and preferably 3.

Among the compounds whose total number of chlorine substituents is 3,preferable compounds are those wherein R¹ and R² are chlorine and R³ andR⁴ are hydrogen; those wherein R¹ and R³ are chlorine and R² and R⁴ arehydrogen; those wherein R² and R³ are chlorine and R¹ and R⁴ arehydrogen; those wherein R² and R⁴ are chlorine and R¹ and R³ arehydrogen; and those wherein R³ and R⁴ are chlorine and R¹ and R² arehydrogen. Particularly preferable are compounds wherein R² and R³ arechlorine and R¹ and R⁴ are hydrogen; and those wherein R² and R⁴ arechlorine and R¹ and R³ are hydrogen. The most preferable are compoundswherein R² and R⁴ are chlorine and R¹and R³ are hydrogen.

Preferably, n is an integer from about 0 to 20, particularly an integerof about 2 to 6, and most preferably 2. When the length of polymethylenechain, n, is within this range, synthesis is facilitated.

The amino acid residue or peptide represented by Z is not particularlylimited so long as the peptide is composed of 100 or less amino acidresidues according to the usual definition of peptide. An amino acidresidue or peptide having about 1 to 50 residues, particularly about 1to 10 residues, more particularly about 1 to 4 residues, furtherparticularly about 1 to 3 residues, is preferable. Excessively longpeptides have increased water solubility and are thus difficult todissolve in organic solvents at the time of measurement and may resultin precipitation in the reaction mixture. Such a problem does not ariseso long as the number of amino acid residues is within theabove-mentioned range.

Specific examples of preferable compounds are those wherein R¹ and R²are chlorine and R³ and R⁴ are hydrogen, n is an integer from 2 to 6,and Z is a peptide having about 1 to 4 amino acid residues: thosewherein R¹ and R³ are chlorine and R² and R⁴ are hydrogen, n is aninteger from 2 to 6, and Z is a peptide having about 1 to 4 amino acidresidues; those wherein R² and R³ are chlorine and R¹ and R⁴ arehydrogen, n is an integer from 2 to 6, and Z is a peptide having about 1to 4 amino acid residues; those wherein R²and R⁴ are chlorine and R¹ andR³ are hydrogen, n is an integer from 2 to 6, and Z is a peptide havingabout 1 to 4 amino acid residues; and those wherein R³ and R⁴ arechlorine and R¹ and R² are hydrogen, n is an integer from 2 to 6, and Zis a peptide having about 1 to 4 amino acid residues.

Among these, preferable compounds are those wherein R² and R³ arechlorine and R¹ and R⁴ are hydrogen, n is an integer from 2 to 6, and Zis a peptide having about 1 to 4 amino acid residues; and those whereinR² and R⁴ are chlorine and R¹ and R³ are hydrogen, n is an integer from2 to 6, and Z is a peptide having about 1 to 4 amino acid residues.

Particularly preferable compounds are those wherein R² and R⁴ arechlorine and R¹ and R³ are hydrogen, n is 5, and Z is an amino acid orpeptide having about 1 to 3 amino acid residues; and those wherein R²and R³ are chlorine and R¹ and R⁴ are hydrogen, n is 2, and Z is anamino acid or peptide having about 1 to 3 amino acid

Use

The compound of formula (1) reacts with an anti-dioxin antibody and thuscan be used as an immunoassay standard for dioxins. Since this compounddoes not have a toxic equivalency factor (TEF), the use of the compoundenables the establishment of an immunoassay system for dioxins thatallows safe measurement.

Production Method

The dioxins immunoassay standard of the invention can be synthesized,for example, by the following method, although the method is not limitedthereto.

The compound of formula (2)

wherein R¹, R², R³ and R⁴ may be the same or different and representchlorine or hydrogen, and n is an integer from 1 to 10, is activated byan activated ester method which comprises reacting the compound withN-hydroxysuccinimide to give an activated ester compound of formula (3)

wherein R¹, R², R³ and R⁴ may be the same or different and representchlorine or hydrogen, and n is an integer from 1 to 10.

The compound of formula (3) is then reacted with an aminogroup-containing compound such as an amino acid or a peptide accordingto an ordinary method to give a compound of formula (1).

The chlorophenol derivative of formula (2) used as the starting compoundcan be synthesized, for example, by the following method. Chlorophenol,potassium carbonate and ethyl 6-bromohexanoate are stirred at 60° C. for16 hours. After completion of the reaction, the reaction mixture isextracted with ethyl acetate. The extract is concentrated under reducedpressure and the residue is dissolved in ethanol. After addition ofsodium hydroxide solution, the mixture is stirred at room temperaturefor 3 hours. After completion of the reaction, the reaction mixture isneutralized with concentrated hydrochloric acid and concentrated underreduced pressure. The resulting mixture is acidified by addition ofconcentrated hydrochloric acid, extracted with ethyl acetate, andrecrystallized to give a chlorophenol derivative compound of formula(2).

(II) Dioxins Immunoassay Kit

The dioxins immunoassay kit of the invention comprises the compound offormula (1) of the invention as a standard for dioxins quantitativedetermination. The anti-dioxin antibody may be prepared by the user ormay be contained in the kit. A kit containing the antibody isconvenient. The anti-dioxin antibody will be described later in anothersection. For competitive immunoassay, the kit may further comprise acompetitive antigen. The competitive antigen will also be describedlater in another section.

The kit may further comprise other items such as a reaction vessel, ablocking agent for masking the free surface of the vessel, a buffer, asecondary antibody for indirect competitive immunoassay and the like.

(III) Dioxin Immunoassay Method

Basic Construction

The method of the invention is an immunoassay for dioxins quantitativedetermination using the compound of formula (1) as a standard. Morespecifically, the method of the invention is an immunoassay forcalculating the concentration of dioxins in a sample, or for calculatingthe concentration of dioxins and further computing the TEQ, the methodusing the compound of formula (1) as a standard.

The dioxins immunoassay of the invention comprises the following steps:

-   (1) reacting a sample with an anti-dioxin antibody to determine the    amount of antigen reacted with the antibody; and-   (2) comparing the amount of antigen reacted with the antibody in    step (1) with that determined by allowing a known concentration of    the compound of formula (1) to react with the anti-dioxin antibody    to compute the concentration of dioxins in the sample.

The dioxins immunoassay of the invention is characterized by using thechlorophenol derivative of formula (1) as a standard for dioxinsimmunoassay and can be carried out using this standard in a similarmanner as usual immunoassay.

The compound of the invention is applicable to any known immunoassaymethod. Examples of such known immunoassay methods include enzymeimmunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay(FIA), and the like. EIA is preferable in view of its simplicity ofmeasurement.

EIA includes competitive immunoassay, non-competitive immunoassay,homogeneous immunoassay, etc. Since dioxins are low molecular weightcompounds, competitive assays may be usually used. Competitiveimmunoassay includes indirect competitive assays in which an antigen isimmobilized on microplate wells, tubes or the like, and directcompetitive assays in which an antibody is immobilized on microplatewells, tubes or the like.

Indirect Competitive Assay

(i) Competitive Antigen

In indirect competitive assays, a dioxin or a complex of dioxin andcarrier protein is used as a competitive antigen immobilized on wells.When the reaction vessel is an untreated vessel made of resin, glass orthe like, a complex of dioxin and carrier protein is preferably usedbecause it is difficult to immobilize a dioxin alone on the surface ofthe vessel. In the case of using a reaction vessel whose surface isactivated by a highly reactive functional group such as an amino groupor a carboxyl group, dioxin alone can be immobilized via the functionalgroup on the vessel. Irrespective of the use of carrier proteins,linkers are preferably attached to dioxins, whereby steric hindrance isalleviated and the reactivity of the competitive antigen with theanti-dioxin antibody improves, resulting in enhanced assay sensitivityfor dioxins in a sample.

Examples of usable dioxins include polychlorinated dibenzo-p-dioxins(PCDDs), polychlorinated dibenzofurans (PCDFs) and coplanarpolychlorinated biphenyls (Co-PCBs). By using a dioxin or a dioxin-likecompound having little similarity to toxic dioxins, reactivity of theanti-dioxin antibody with the competitive antigen is less than with thedioxins in a sample, resulting in improved detection sensitivity of thedioxins concentration in the sample.

The carrier protein is not particularly limited and any known carrierproteins can be used. Examples of carrier proteins include KLM (Keyholelimpet hemocyanin), bovine serum albumin (BSA) and the like.

The linker is preferably one that does not sterically hinder bindingwith the antibody nor adversely affect solubility in the reactionprocess. Examples of usable linkers include polymethylene chains and thelike. The linker is disposed between the dioxins or dioxins-likecompound and the container or between the carrier protein complex of thedioxin or dioxin-like compound and the container.

Highly sensitive quantitative determination of dioxins can be achievedby using the chlorophenol derivative of formula (1) of the invention asa competitive antigen. It is particularly preferable that thecompetitive antigen be the same compound as the standard for thecalibration curve preparation. In this case, an antigen to beimmobilized can be produced by replacing the amino acid or peptidemoiety at an end of the substituted compound of the standard representedby formula (1) with a carrier protein such as BSA.

(ii) Anti-dioxin Antibody

The antibody used in EIA can be prepared by a known method comprisinghaptenizing a dioxin such as polychlorinated dibenzo-p-dioxin (PCDD),polychlorinated dibenzofuran (PCDF) or coplanar polychlorinated biphenyl(Co-PCB), conjugating the hapten to a carrier protein such as BSA andimmunizing a mammal using the conjugate as an immunization antigen (KunChae, et al., J. Agric. Food., 25, 1207–1209 (1977); Simona G. Merica,et al., Can. J. Chem., 73, 826–834 (1995)).

The antibody may be monoclonal or polyclonal and is not especiallylimited. The use of a monoclonal antibody is preferable in view ofantibody homogeneity and unlikelihood of lot-to-lot differences inantibody production. The monoclonal antibody is an antibody obtainedfrom single antibody-producing cells that are prepared by cloning ahybridoma produced by the fusion of a haptenized dioxins-immunized mousespleen cell with a tumor cell. Any monoclonal antibody that recognizesdioxins can be used.

The indirect competitive assay can be carried out, for example, in thefollowing manner. First, the competitive antigen is immobilized on thesurface of the wells of a reaction vessel such as a microplate. Then theportion of the surface of the wells to which the antigen is not attachedis blocked with a commercially available blocking agent such as bovineserum albumin, casein or the like. A sample and a primary antibody(anti-dioxin antibody) is added to the wells, and the sample and theimmobilized antigen are allowed to competitively interact with theantibody. Antibodies not bound to the immobilized antigen are removed bywashing. Then a secondary antibody, for example, an enzyme-labeledantibody prepared by labeling a goat anti-mouse immunoglobulin antibodywith peroxidase (HRP), alkaline phosphatase (ALP) or like enzyme, isadded to the wells and allowed to bind to the primary antibody bindingto the immobilized antigen. After the resulting antibody conjugate iswashed with buffer several times, the substrate for the labeled enzymeis added and absorbance of the colored enzymatic reaction product ismeasured. When the enzyme is peroxidase, hydrogen peroxide may be usedas a substrate, and o-phenylenediamine or tetramethylbenzidine may beused as the coloring agent. When the enzyme is alkaline phosphatase,p-nitrophenylphosphoric acid is usually used as a substrate.

In the above competitive assay, the percentage of inhibition by a sampleis obtained by determining the percent absorbance decrease by additionof a test sample relative to the absorbance of the sample-free reactionmixture. Using the compound of formula (1) as a dioxin standard, acompetitive reaction is carried out in the same manner except usingseveral known concentrations of the standard solution in place of thesample to prepare a calibration curve relating the standard solutionconcentrations and the percentage of inhibition. The dioxinsconcentration in the sample is calculated in terms of standard solutionconcentration by comparing the calibration curve and the percentage ofinhibition.

Direct Competitive Immunoassay

In direct competitive immunoassay, the anti-dioxin antibody isimmobilized on the surface of the wells of a reaction vessel and theportion of the surface of the wells to which the antigen is not boundare blocked in the same manner as above. By adding a competitiveenzyme-labeled antibody and a test sample to the wells, the sample andthe enzyme-labeled antigen are allowed to competitively interact withthe immobilized antibody. The enzyme-labeled antigen not bound to theantibody is then removed by washing and the substrate for the labelingenzyme is added to determine the absorbance of the reaction product.

The enzyme-labeled antigen can be prepared by linking peroxidase,alkaline phosphatase or like enzyme to the same dioxin or dioxin-likecompound used as a competitive antigen in indirect competitiveimmunoassay. When using an antigen prepared by replacing the amino acidor peptide moiety at an end of the compound of formula (1) with anenzyme such as peroxidase or alkaline phosphatase, sensitivity isenhanced.

When RIA is carried out instead of EIA, a labeled antigen can beprepared by labeling the dioxins or dioxins-like compound with anisotope. When FIA is carried out, a labeled antigen can be prepared byattaching a fluorescent substance such as rhodamine or achemiluminescent substance to the dioxins or dioxins-like compound.

Test Sample

The sample type is not specifically limited and may include, forexample, environmental samples collected from the environment,biological samples, foods and like various products, and dioxinsolutions prepared for experiments. The method of the invention isespecially suitable for environmental samples for use as test samples.Examples of environmental samples include air; exhaust gases fromautomobiles, machines and devices, factories, and the like; soils;river, lake, harbor or port waters; combustion or fly ashes and thelike. Biological samples include mothers' milk, blood and urine.

TEQ Calculation Method

The test sample may be directly subjected to immunoassay or may bepretreated to extract a fraction containing large amounts of dioxintherefrom. Performing a pretreatment is preferable. Examples ofpretreatment methods are given below, but are not limiting.

(i) Exhaust Gas Sample

A gas sample of A Nm³ is collected and the substances contained thereinare extracted with toluene. The extract is measured to 20 ml and it isreferred to as a crude extract. 10 ml of the extract is treated withsulfuric acid until the sulfuric acid layer becomes colorless. Thetreated extract is dissolved in hexane and cleaned up by applying it toa multilayered silica gel column comprising layers of 1 g of sodiumsulfate, 1 g of 10% silver nitrate silica gel and 3 g of silica gel with200 ml of hexane. 1 ml of a DMSO solution containing this extract issubjected to immunoassay.

The obtained dioxins concentration in terms of the standard, hereinaftersimply referred to as “Standard dioxins concentration”, is inserted intothe following equation to calculate toxicity equivalent (TEQ):TEQ(ng−TEQ/Nm³)=0.0922×Standard dioxins concentration (μg/Nm³)^(0.8474)

In the above formula, 0.0922 and 0.8474 are coefficients in thecorrelation equation between the dioxins concentration in exhaust gasesdetermined by the method of this invention in the Example and thedioxins TEQ obtained by the official method. These are constantsassigned to each type of sample. Unit conversion of the Standard dioxinsconcentration can be done by the following formula:Standard dioxins concentration (μg/N m³)=Standard dioxins concentration(ng/ml)×1/10×20×1/A×1/1000(ii) Fly Ash

Fly ashes (B g) are collected and the compounds contained therein areextracted with toluene. The extract is measured to 20 ml and it isreferred to as a crude extract. 1 ml of the extract is measured out andcleaned up. 2 ml of a DMSO solution containing this extract is subjectedto immunoassay.

The obtained Standard dioxins concentration is inserted into thefollowing equation to calculate toxicity equivalent (TEQ):TEQ(ng−TEQ/g)=0.0038×[Standard dioxins concentration (μg/g)]0.9796

In the above formula, the values 0.0038 and 0.9796 are coefficients inthe correlation equation between the dioxins concentration in fly ashesdetermined by the method of this invention in the Example and the dioxinTEQ obtained by the official method. These values are constants assignedto each type of sample.

Unit conversion of the Standard dioxins concentration can be done by thefollowing formula:Standard dioxins concentration (μg/g)=Standard dioxins concentration(ng/ml)×2/1×20×1/B×1/1000(iii) Soil

Soil (B g) is collected and the compounds contained therein extractedwith toluene. The extract is measured to 20 ml and it is referred to asa crude extract. 1 ml of the crude extract is measured out and cleanedup. 2 ml of a DMSO solution containing this extract is subjected toimmunoassay. The obtained Standard dioxins concentration is insertedinto the following equation to calculate toxicity equivalent (TEQ):TEQ(ng−TEQ/g)=9.4553×[Standard dioxins concentration (μg/g)]^(0.9352)

In the above formula, the values 9.4553 and 0.9352 are coefficients inthe correlation equation between the dioxins concentration in fly ashesdetermined by the method of this invention in the Example and thedioxins TEQ obtained by the official method. These values are constantsassigned to each type of sample. Unit conversion of the Standard dioxinsconcentration can be done in the same manner as in fly ashes.

EXAMPLES

Examples are given below to illustrate the invention in more detail. Thescope of the invention is not limited to these examples.

Example 1

Preparation of Dioxins Immunoreaction Assay Standard

Of the compounds of formula (1) of the invention, a compound in which R²and R⁴ are chlorine and R¹ and R³ are hydrogen was synthesized by thefollowing method. The synthetic procedure is described with reference toFIG. 1.

Under argon gas, 15.0 g (76.0 mmol) of 2,4,5-trichlorophenol (1)(commercially available product), 18.6 g (83.6 mmol) of ethyl6-bromohexanoate, 12.60 g (91.2 mmol) of potassium carbonate, and 150 mlof anhydrous dimethylformamide were mixed and heated with stirring at60° C. overnight. After completion of the reaction, the reaction mixturewas cooled to room temperature. After addition of 450 ml of water, thereaction mixture was extracted with 225 ml of ethyl acetate twice. Theextract was dried over magnesium sulfate, the desiccant was filtered offand the organic layer was concentrated to give 30.7 g of a pale yellowoil as a crude product. The crude product was purified by silica gelcolumn chromatography (silica gel: 450 g, eluant: ethylacetate/n-hexane=1:15) to give 26.5 g of ethyl6-(2,4,5-trichlorophenoxy)hexanoate (2) as a transparent oil (100%yield).

The ethyl 6-(2,4,5-trichlorophenoxy)hexanoate was dissolved in 200 ml ofethanol and then 200 ml of 2N aqueous sodium hydroxide solution wasadded dropwise with ice-cooling and stirred at room temperature for 3hours. After completion of the reaction, the reaction mixture wasneutralized with 70 ml of concentrated hydrochloric acid andconcentrated to half its original volume. The concentrated reactionmixture was acidified by addition of 5 ml of concentrated hydrochloricacid and extracted with 100 ml of ethyl acetate once and with 150 ml ofethyl acetate twice. The organic layer was washed with 200 ml of waterand then with 200 ml of saturated aqueous sodium chloride solution anddried over magnesium sulfate. After filtering off the desiccant, theorganic layer was concentrated to give 23.3 g of a white solid as acrude product. After addition of 25 ml of isopropyl ether and 50 ml ofn-hexane, the crude product was recrystallized. The precipitatedcrystals were collected by filtration and washed with isopropylether/n-hexane (isopropyl ether/n-hexane=1:3) to give6-(2,4,5-trichlorophenoxy)hexanoic acid (3) as white crystals (88.0%yield). 18.2 g (58.4 mmol) of 6-(2,4,5-trichlorophenoxy)hexanoic acid(3) was dissolved in 180 ml of methylene chloride and then 12.7 g (70.1mmol) of 1-ethyl-3-(3′-diethylaminopropyl)carbodiimide hydrochloride and8.07 g (70.1 mmol) of N-hydroxysuccinimide were added, followed bystirring at room temperature overnight. After completion of thereaction, the reaction mixture was added to 1250 ml of THF. The organiclayer was successively washed with 360 ml of water, 540 ml of saturatedaqueous sodium hydrogen carbonate solution and 540 ml of water and driedover sodium sulfate. After filtering off the desiccant, the organiclayer was concentrated to give 21.9 g of a white solid as a crudeproduct. The crude product was purified by silica gel columnchromatography (using 400 g of silica gel and methylene chloride aseluant for the first chromatographic step; using 330 g of silica gel andmethylene chloride as eluant for the second chromatographic step) togive 9.73 g of succinimidyl 6-(2,4,5-trichlorophenoxyl)hexanoate (4) asa white solid (40.7% yield).

20 mg of succinimidyl 6-(2,4,5-trichlorophenoxyl)hexanoate (4) wasdissolved in 100 ml of dimethyl sulfoxide. Then 100 ml of 50 mM aqueousglycylglycine solution was gradually added and stirred at roomtemperature for 3 hours to give an immunoassay standard solution fordioxins.

A compound of formula (1) in which R² and R³ are chlorine and R¹ and R⁴are hydrogen was synthesized in the same manner as above except that2,4,6-trichlorophenol (commercially available product) was used as astarting compound in place of 2,4,5-trichlorophenol.

In addition, a compound of formula (1) in which R¹ and R⁴ are chlorineand R² and R³ are hydrogen was synthesized in the same manner as aboveexcept that 3,4,5-trichlorophenol (commercially available product) wasused as a starting material in place of 2,4,5-trichlorophenol.

Example 2

Preparation of Competitive Assay Antigen

A competitive assay antigen was prepared using succinimidyl6-(2,4,5-trichlorophenoxyl)hexanoate (4) obtained in Example 1 andbovine serum albumin (BSA). More specifically, first, 545.5 μl ofdimethyl sulfoxide was added to 1 ml of a bovine serum albumin (BSA)solution in 50 mM phosphate buffer (pH 8.0) (corresponding to 15 mg(2.27×10⁻⁷ mol of BSA)) with stirring while being cooled with ice. Then454.5 μl (40 equivalents (9.09×10⁻⁶ mol)) of succinimidyl6-(2,4,5-trichlorophenoxyl)hexanoate (BB2-4) was added dropwise andallowed to react at room temperature for 1 hour. After the reaction, 4Lof PBS was dialyzed to give a competitive assay antigen.

Example 3

Preparation of Exhaust Gas Sample and Determination of Dioxins TEQ bythe Official Method

Exhaust gas emitted from a waste incineration facilities was collectedand the dioxins concentration of the sample was determined using a highperformance gas chromatography-mass spectrometer (GC-MS) according toJIS K0311 (the official method). In addition, about 3 Nm³ of exhaust gaswas sucked from the flue of a city waste incineration facilities using agas sampling device and the sample was extracted with an organic solventsuch as toluene or dichloromethane, selected in accordance with the formsuch as filter paper, resin or absorbent liquid. These extracts werecombined and concentrated to 20 ml to give a crude extract.

1 ml was measured out of the crude extract and purified by sulfuric acidtreatment, multilayered silica gel chromatography, and activated carboncolumn chromatography. The concentrations of dioxin isomers weredetermined using a gas chromatography-mass spectrometer (product ofMicromass).

The concentrations of the dioxin isomers were then multiplied by theirTEFs to obtain the dioxins TEQ of the exhaust gas sample.

In the meantime, another 1 ml of the crude extract was measured out andpurified by sulfuric acid treatment and multilayered silica gelchromatography. After evaporating organic solvent, the residue wasdissolved in 2 ml of dimethyl sulfoxide (DMSO) and used as an EIAevaluation sample.

Example 4

TEQ Determination of Exhaust Gas Sample Using Anti-dioxins MonoclonalAntibody by Indirect Competitive Assay

A competitive assay antigen prepared in Example 2 was dissolved in PBSto a concentration of about 1 μg/ml, and 100 μl of this solution waspipetted into each well. The plate was hermetically sealed and allowedto stand at 4° C. for 18 hours to solidify the solution. After removalof the antigen solution, the plate was washed with 0.05% Tween20-containing PBS three times and 300 μl of a 5-fold diluted blockingsolution (product of Nacalai Tesque Co.) was pipetted into each well.The plate was hermetically sealed and allowed to stand at 4° C.overnight for blocking, thus giving a immunoassay plate for dioxins.Using this plate, dioxins immunoassay was performed in the followingmanner.

The dioxins standard prepared in Example 1 was diluted with 50% DMSOcontaining 0.01% Triton X100 to produce a dilution series ranging from 0to 0.2 μg/ml. The exhaust gas sample prepared in Example 3 was dissolvedin DMSO and diluted with 50% DMSO containing 0.01% Triton X100 to givean assay sample.

In the meantime, a hybridoma that produces an anti-dioxins monoclonalantibody was cultured in a serum-free medium in a CO₂ incubator at 37°C. for 1 week or more and the culture supernatant thus obtained waspurified by affinity chromatography using a protein A column anddialyzed in PBS to provide an anti-dioxins monoclonal antibody.

50 μg of each of the dilution series of the dioxins standard and theexhaust gas sample were added to the dioxins assay plate and 50 μl of ananti-dioxins monochronal antibody solution that had been diluted withPBS containing 0.2% BSA to a concentration of 0.1 μg/ml was added toeach well and allowed to react at 4° C. for 1 hour. After the reaction,the solution added to each well was removed and the plate was washedwith PBS containing 0.005% Tween 20 three times. Then 100 μl of a goatanti-mouse IgG(H+L) HRP labeled antibody (obtained by affinitypurification, product of DAKO) diluted 2000-fold with PBS containing0.2% BSA was pipetted into each well. The plate was allowed to stand atroom temperature for 1 hour and washed with PBS containing 0.05% Tween20 three times. Then 100 μl of HRP substrate TMB (product of BioFX) waspipetted into each well and allowed to stand at room temperature for 30minutes. After addition of 100 μl of 0.5M sulfuric acid solution to eachwell, absorbance was measured at a wavelength of 455 nm (655 nm forcontrol) using a microtiter spectrophotometer.

A standard curve (calibration curve) was prepared from absorbance dataobtained using the dioxins standard. Absorbance data obtained using theexhaust gas sample was compared and fitted to the calibration curve todetermine the amount of dioxins in the sample in terms of standardsubstance concentration.

FIG. 2 shows the obtained standard curve. In FIG. 2, B representsabsorbance in the presence of the standard, and B₀ represents absorbancein the absence of the standard.

FIG. 3 shows the correlation between dioxins TEQ obtained byquantitative determination using GC-MS in Example 3 and dioxinsconcentration in the sample obtained by performing immunoassay using thestandard of the invention in Example 4. As is clear from FIG. 3, thereis a good correlation between the measurement obtained by immunoassay ofexhaust gas samples using the immunoassay standard of the invention anddioxins TEQ calculated by the official instrumental analysis method(R²=0.985).

Example 5

Preparation of Fly Ash Sample and Dioxins TEQ Determination by theOfficial Method

TEQ in fly ash samples was calculated from the results of GC-MS in thesame manner as in Example 3.

Example 6

TEQ Determination of Fly Ash Sample Using Anti-dioxins MonoclonalAntibody by Indirect Competitive Assay

Dioxin TEQ of fly ash samples was determined by EIA in the same manneras in Example 4.

FIG. 4 shows the correlation between dioxins TEQ obtained byquantitative determination using GC-MS in Example 5 and dioxinsconcentration of the sample obtained by performing immunoassay using thestandard of the invention in Example 6. As is clear from FIG. 4, thereis a good correlation between the measurement obtained by immunoassay ofthe fly ash samples using the immunoassay standard of the invention anddioxins TEQ calculated by the official instrumental analysis method(R²=0.990).

Example 7

Preparation of Soil Sample and Determination of Dioxins TEQ by theOfficial Method

TEQ in soil samples was calculated from the results of GC-MS analysis inthe same manner as in Example 3.

Example 8

TEQ Determination of Soil Sample Using Anti-Dioxins Monoclonal Antibodyby Indirect Competitive Assay

Dioxin TEQ of soil samples was determined by EIA in the same manner asin Example 4.

FIG. 5 shows the correlation between dioxins TEQ obtained byquantitative determination using GC-MS in Example 7 and dioxinsconcentration of the sample obtained by performing immunoassay using thestandard of the invention in Example 8. As is clear from FIG. 5, thereis a good correlation between the measurement obtained by immunoassay ofthe soil samples using the immunoassay standard of the invention anddioxins TEQ calculated by the official instrumental analysis method(R²=0.992).

In the above, the correlation with the official method was discussedregarding a compound of formula (1) wherein R² and R⁴ are chlorine andR¹ and R³ are hydrogen. Using a compound of formula (1) wherein R² andR³ are chlorine and R¹ and R⁴ are hydrogen and a compound of formula (1)wherein R¹ and R⁴ are chlorine and R² and R³ are hydrogen, the sameprocedure as in Examples 2 to 8 was repeated. There was also a highcorrelation with these compounds to the official method for dioxins TEQ.

INDUSTRIAL APPLICABILITY

The compound and method of the invention are suitable for quantitativedetermination of dioxins in environmental samples such as soil, air,exhaust gases, lake and river waters; biological samples such asmothers' milk, blood and urine; and products such as foods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a synthetic scheme for the dioxins immunoassay standard ofExample 1.

FIG. 2 shows a standard curve obtained using an embodiment of thedioxins immunoassay standard of the invention.

FIG. 3 shows the correlation between dioxins TEQ of exhaust gas obtainedby gas chromatography-mass spectrometry and dioxins concentration of theexhaust gas obtained by performing immunoassay using the dioxinsimmunoassay standard of the invention.

FIG. 4 shows the correlation between dioxins TEQ of fly ash obtained bygas chromatography-mass spectrometry and dioxins concentration of thefly ash obtained by performing immunoassay using the dioxins immunoassaystandard of the invention.

FIG. 5 shows the correlation between dioxins TEQ of soil obtained by gaschromatography-mass spectrometry and dioxins concentration of the soilobtained by performing immunoassay using the dioxins immunoassaystandard of the invention.

1. An immunoassay method for determining the concentration of dioxins ina sample, the method comprising the following steps: 1) allowing targetdioxins in the sample and a competitive antigen to competitively reactwith a primary anti-dioxin antibody capable of binding to the targetdioxins, and determining the amount of competitive antigen-antibodycomplex from a label incorporated into a secondary antibody binding tothe primary antibody; 2) allowing the competitive antigen and a compoundof formula (1) of known concentration

wherein R¹, R², R³ and R⁴ may be the same or different and eachrepresents chlorine or hydrogen, n is an integer from 1 to 10, and Zrepresents 1 to 100 amino acid residues to competitively react with theprimary anti-dioxin antibody, and determining the amount of competitiveantigen-antibody complex from a label incorporated into a secondaryantibody binding to the primary antibody; 3) preparing a calibrationcurve using the amount of competitive antigen-antibody complexdetermined in step 2); and 4) comparing the amount of competitiveantigen-antibody complex determined in step 1) with the calibrationcurve prepared in step 3).
 2. The immunoassay method according to claim1, wherein the competitive antigen is a compound of formula (1) whereinZ is a carrier protein.
 3. The immunoassay method according to claim 1,wherein the label is an enzyme, a radioactive substance, or afluorescent substance.
 4. The immunoassay method according to claim 1,wherein in formula (1), R² and R⁴ are chlorine, R¹ and R³ are hydrogen,n is 5, and Z represents 1 to 3 amino acid residues.
 5. The immunoassaymethod according to claim 1, wherein in formula (1), R² and R³ arechlorine, R¹ and R⁴ are hydrogen, n is 5, and Z represents 1 to 4 aminoacid residues.
 6. An immunoassay method for determining theconcentration of dioxins in a sample, the method comprising thefollowing steps: 1) allowing target dioxins in the sample and a labeledcompetitive antigen to competitively react with a primary anti-dioxinantibody capable of binding to the target dioxins, and determining theamount of competitive antigen-antibody complex from a label incorporatedinto the competitive antigen; 2) allowing the competitive antigen and acompound of formula (1) of known concentration

wherein R¹, R², R³ and R⁴ may be the same or different and eachrepresents chlorine or hydrogen, n is an integer from 1 to 10, and Zrepresents 1 to 100 amino acid residues to competitively react with theprimary anti-dioxin antibody, and determining the amount of competitiveantigen-antibody complex from a label incorporated into the competitiveantigen; 3) preparing a calibration curve using the amount ofcompetitive antigen-antibody complex determined in step 2); and 4)comparing the amount of competitive antigen-antibody complex determinedin step 1) with the calibration curve prepared in step 3).
 7. Theimmunoassay method according to claim 6, wherein the competitive antigenis a compound of formula (1) wherein Z is a carrier protein.
 8. Theimmunoassay method according to claim 6, wherein the label is an enzyme,a radioactive substance or a fluorescent substance.
 9. The immunoassaymethod according to claim 6, wherein in formula (1), R² and R⁴ arechlorine, R¹ and R³ are hydrogen, n is 5, and Z represents 1 to 3 aminoacid residues.
 10. The immunoassay method according to claim 6, whereinin formula (1), R² and R³ are chlorine, R¹ and R⁴ are hydrogen, n is 5,and Z represents 1 to 4 amino acid residues.
 11. A method of evaluatingthe toxic equivalent (TEQ) of dioxins in a sample, the method comprisingthe following steps: 1) allowing target dioxins in the sample and acompetitive antigen to competitively react with a primary anti-dioxinantibody capable of binding to the target dioxins, and determining theamount of competitive antigen-antibody complex from a label incorporatedinto a secondary antibody binding to the primary antibody; 2) allowingthe competitive antigen and a compound of formula (1) of knownconcentration

wherein R¹, R², R³ and R⁴ may be the same or different and eachrepresents chlorine or hydrogen, n is an integer from 1 to 10, and Zrepresents 1 to 100 amino acid residues to competitively react with theprimary anti-dioxin antibody, and determining the amount of competitiveantigen-antibody complex from a label incorporated into a secondaryantibody binding to the primary antibody; 3) preparing a calibrationcurve using the amount of competitive antigen-antibody complexdetermined in step 2); 4) comparing the amount of competitiveantigen-antibody complex determined in step 1) with the calibrationcurve prepared in step 3); and 5) calculating the TEQ of dioxins in asample.
 12. The method according to claim 11, wherein the competitiveantigen is a compound of formula (1) wherein Z is a carrier protein. 13.The method according to claim 11, wherein the label is an enzyme, aradioactive substance or a fluorescent substance.
 14. The methodaccording to claim 11, wherein in formula (1), R² and R⁴ are chlorine,R¹ and R³ are hydrogen, n is 5, and Z represents 1 to 3 amino acidresidues.
 15. The method according to claim 11, wherein in formula (1),R² and R³ are chlorine, R¹ and R⁴ are hydrogen, n is 5, and Z represents1 to 4 amino acid residues.
 16. A method of evaluating the toxicequivalent (TEQ) of dioxins in a sample, the method comprising thefollowing steps: 1) allowing target dioxins in the sample and a labeledcompetitive antigen to competitively react with a primary anti-dioxinantibody capable of binding to the target dioxins, and determining theamount of competitive antigen-antibody complex from a label incorporatedinto the competitive antigen; 2) allowing the competitive antigen and acompound of formula (1) of known concentration

wherein R¹, R², R³ and R⁴ may be the same or different and eachrepresents chlorine or hydrogen, n is an integer from 1 to 10, and Zrepresents 1 to 100 amino acid residues to competitively react with theprimary anti-dioxin antibody, and determining the amount of competitiveantigen-antibody complex from a label incorporated into the competitiveantigen; 3) preparing a calibration curve using the amount ofcompetitive antigen-antibody complex determined in step 2); 4) comparingthe amount of competitive antigen-antibody complex determined in step 1)with the calibration curve prepared in step 3); and 5) calculating theTEQ of dioxins in a sample.
 17. The method according to claim 16,wherein the competitive antigen is a compound of formula (1) wherein Zis a carrier protein.
 18. The method according to claim 16, wherein thelabel is an enzyme, a radioactive substance or a fluorescent substance.19. The method according to claim 16, wherein in formula (1), R² and R⁴are chlorine, R¹ and R³ are hydrogen, n is 5, and Z represents 1 to 3amino acid residues.
 20. The method according to claim 16, wherein informula (1), R² and R³ are chlorine, R¹ and R⁴ are hydrogen, n is 5, andZ represents 1 to 4 amino acid residues.
 21. The method according toclaim 1, wherein in formula (1), R² and R⁴ are chlorine, R¹ and R³ arehydrogen, n is 5, and Z represents glycylglycine.
 22. The methodaccording to claim 1, wherein in formula (1) used in step 2), R² and R⁴are chlorine, R¹ and R³ are hydrogen, n is 5, and Z representsglycylglycine and the competitive antigen is a compound of formula (1)wherein R¹, R², R³, and R⁴ are, independently, a chlorine or hydrogenatom, n is an integer from 1 to 10, and Z is a carrier protein.
 23. Themethod according to claim 6, wherein in formula (1), R² and R⁴ arechlorine, R¹ and R³ are hydrogen, n is 5, and Z representsglycylglycine.
 24. The method according to claim 6, wherein in formula(1) used in step 2), R² and R⁴ are chlorine, R¹ and R³ are hydrogen, nis 5, and Z represents glycylglycine and the competitive antigen is acompound of formula (1) wherein R¹, R², R³, and R⁴ are, independently, achlorine or hydrogen atom, n is an integer from 1 to 10, and Z is acarrier protein.
 25. The method according to claim 11, wherein informula (1), R² and R⁴ are chlorine, R¹ and R³ are hydrogen, n is 5, andZ represents glycylglycine.
 26. The method according to claim 11,wherein in formula (1) used in step 2), R² and R⁴ are chlorine, R¹ andR³ are hydrogen, n is 5, and Z represents glycylglycine and thecompetitive antigen is a compound of formula (1) wherein R¹, R², R³, andR⁴ are, independently, a chlorine or hydrogen atom, n is an integer from1 to 10, and Z is a carrier protein.
 27. The method according to claim16, wherein in formula (1), R² and R⁴ are chlorine, R¹ and R³ arehydrogen, n is 5, and Z represents glycylglycine.
 28. The methodaccording to claim 16, wherein in formula (1) used in step 2), R² and R⁴are chlorine, R¹ and R³ are hydrogen, n is 5, and Z representsglycylglycine and the competitive antigen is a compound of formula (1)wherein R¹, R², R³, and R⁴ are, independently, a chlorine or hydrogenatom, n is an integer from 1 to 10, and Z is a carrier protein.